Fluorescence detection methods for detecting an intensity of fluorescence which is emitted from a fluorescently-labeled biological sample have conventionally been known. According to the fluorescence detection methods, separation or identification of a gene sequence, a level of gene expression, or a protein, or evaluation of a molecular weight or a property can be achieved by detecting the fluorescence intensity.
For example, μTAS-immunoassay system (Micro Total Analysis System—Enzyme Linked Immuno-Sorbent Assey (ELISA) has been known. This uses a microchip which is provided with a micro flow channel having very small width and depth. An electrophoretic fluid (buffer fluid) is injected into the flow channel of the microchip, and a biological sample is injected into the micro flow channel. Then, a high voltage (electrophoretic voltage) is applied to induce electrophoresis, and an obtained substance is exposed to excitation light at a measurement area of the microchip to excite the fluorescent label. Then, the intensity of the thus emitted fluorescence is detected to analyze the substance derived from a living organism.
A further example of a microchip-based analysis device is a microarray, and a microarray analysis system has attracted attention. For example, a specific binding substance which can bind specifically to a substance derived from a living organism, such as a hormone, a tumor marker, an enzyme, an antibody, an antigen, an abzyme, any of other kind of proteins, a nucleic acid, a cDNA, a DNA or a RNA, and which has a known base sequence, a known base length, a known composition, and the like, is dropped, using a spotter device, at different positions on a surface of a carrier, such as a slide glass plate or a membrane filter, of the microarray to form a number of independent spots. Then, a fluorescently-labeled substance, which is a substance collected from a living organism through extraction, isolation, or the like, or derived from a living organism and subjected to a chemical treatment or chemical modification, such as a hormone, a tumor marker, an enzyme, an antibody, an antigen, an abzyme, any of other proteins, a nucleic acid, a cDNA, a DNA or a mRNA, is bound specifically to the specific bonding substance on the microarray through hybridization, or the like. Then, excitation light is applied to the microarray, and the intensity of fluorescence emitted from the fluorescent label is photoelectrically detected to analyze the substance derived from a living organism.
In the above-described analysis technique, however, the fluorescence emitted from the fluorescently-labeled biological sample fed to the microchip when it is exposed to the excitation light includes not only the fluorescence emitted from the fluorescently-labeled biological sample but also fluorescence emitted from the microchip itself. The fluorescence emitted from the microchip acts as background and lowers the accuracy, i.e., the S/N ratio, of the detection of the fluorescence intensity.
In a fluorescence detection method proposed in Japanese Unexamined Patent Publication No. 2002-181708, the fluorescence emitted from the fluorescently-labeled biological sample is detected after the intensity of the fluorescence emitted from the microchip itself has sufficiently attenuated. In this method, even if the fluorescence emitted from the microchip itself acts as the background, its intensity is small. Thus, influence of the fluorescence emitted from the microchip itself is reduced, thereby minimizing lowering of the accuracy of detection, i.e., the S/N ratio.
In a method proposed in Japanese Unexamined Patent Publication No. 2003-084002, the background is reduced by treating the microchip with a blocking agent containing a quencher.
In recent years, however, in order to reduce the cost of the microchip, use of a resin material, in place of conventional silica glass, has been proposed as a material forming the microchip. When the excitation light is applied to a resin microchip, the detected fluorescence emitted from the microchip itself is more intense than that from a silica glass microchip. Since the resin microchips are thicker than the silica glass microchips to ensure flatness after the shape forming thereof, more intense fluorescence is emitted therefrom. Therefore, it takes longer time for the intensity of the fluorescence emitted from the microchip itself to sufficiently attenuate than that from the conventional silica glass microchip, and therefore the fluorescence emitted from the microchip acts as the background for longer time. Thus, the influence thereof exerted on the accuracy of detection of the fluorescence intensity of the biological sample is higher. Further, in order to improve productivity of fluorescence detection apparatuses, there is a demand for reduction of time taken for detecting the intensity of the fluorescence emitted from the fluorescently-labeled biological sample.
In the technique disclosed in Japanese Unexamined Patent Publication No. 2002-181708, although the influence of the fluorescence emitted from the microchip itself acting as the background on the accuracy of detection of the fluorescence intensity can be reduced, a time taken for detecting the fluorescence intensity from the biological sample cannot be reduced.
In the technique disclosed in Japanese Unexamined Patent Publication No. 2003-084002, it is necessary to treat the microchip with the blocking agent containing a quencher, and this increases the cost of the microchip.